Project Summary Coccidioidomycosis, also known as Valley fever affects residents in the southwestern United States, northern Mexico and scattered areas of South America. An estimated 150,000 people in the United States become infected with Coccidioides annually. There is an urgently unmet need to develop novel and better chemotherapies against Coccidioides infection. We have developed a phenotypical screening method by utilizing imaging flow cytometry (IFC) to assess fungal cytological profile (FCP) that covers ~300 parameters, including morphology, cell wall integrity, membrane permeability, growth inhibition, etc. We first acquire FCPs of fungal cells incubated separately with fluconazole, amphotericin B and caspofungin compared to cells in medium alone to establish a FCP database for each targeted fungal species. C. immitis and C. posadasii have unique dimorphic life cycles that form mycelia in vitro and spherules (not yeast) in vivo. Infection usually occurs by inhalation of the airborne spores that first convert to spherule initials (<15m) in the lungs. Subsequently, spherule initials grow into large multinucleate spherules (15-120?m) that reproduce by forming endospores. We found that spherules have significantly elevated MIC50 values for clinical drugs compared to spherule initials and hyphae. Thus we propose to apply our newly development IFC to screen well characterized repurposing drug libraries to identify compounds that can alter FCPs and inhibit growth of spherules as outlined in 2 Specific Aims. Aim 1. To complete high-content screening of REPO library to identify drugs that can alter spherule FCP and inhibit growth of Coccidioides. As proof-of-concept, the Prestwick library (1280 off- patented drugs) were screened to identify compounds against Cryptococcus neoformans. We have identified 7 compounds that can also inhibit growth of Coccidioides spherules with various MIC50 (0.3-9.7g/mL). We then applied this IFC method to screen the Pathogen-box library (400 compounds) against Coccidioides spherules and we have identified additional 7 hits for further characterization. We will continue to screen the Repurposing Hub drug library (REPO; 5440 unique compounds). MIC50 of the hits will be assessed by growth inhibition assays against spherules for down-selecting 10 candidates for further assessment. Aim 2. To characterize and prioritize compounds for advancing anti-Coccidioides treatment through in vitro studies. We will assess in vitro antifungal activity of the best 10 compounds against a panel Coccidioides isolates including Flu and AmB resistant strains. The top 5 compounds with overall best MICs will be advanced to the subsequent assays including synergy effect, expanded antifungal activity, cytochrome P450 interaction and cytotoxicity. The goal is to identify at least 3 lead compounds that have the best MIC50, toxicity index (CC50/MIC50) and no considerable anti-synergistic effect with fluconazole and amphotericin B for further preclinical development.